It is well-established that the stem cell's niche is crucial for its maintenance, and recent studies suggest that instructive signals from the microenvironment are able to induce stem cell features in other non-stem cells. De-differentiation, the progression of cells from a more to a less differentiated state, is a key principle in tissue regeneration in lower vertebrates and involves the use of existing, terminally differentiated cells rather than stem cells. In mammals, dedifferentiation has been demonstrated in some organs such as liver and pancreas where tissue regeneration occurs without the aid of identifiable stem cells. However, the cellular and molecular mechanisms that control de-differentiation and reprogramming are little understood. We recently came upon unexpected results when cultures of mouse epidermal keratinocytes that were, by all measures, terminally differentiated were able to reform a fully functional, multi-lineage, hair-bearing epidermis when implanted into a full thickness wound bed in mice. It appeared that terminally differentiated cells were de-differentiated to stem cells. Although we would like to propose this model for exploring mechanisms of de-differentiation and reprogramming, we feel such a application would be premature without experimental evidence that this phenomenon occurs in vivo, in intact skin. This Exploratory Grant project is designed to provide direct evidence for reversion of terminally differentiated keratinocytes to stem cells in vivo and to develop key resources for studying the mechanism of de-differentiation. Experiments are proposed in which double transgenic mice are constructed that incorporate the Cre- LoxP system and express a reporter gene only in terminally differentiated keratinocytes. The constructs are such that if and when terminally differentiated cells do undergo reprogramming to stem cells, reporter gene expression will no longer be confined to terminally differentiated layers of the skin but will be evident in the basal, replicative compartment. Although this application outlines a specific question, implications of this work address broad issues such as - To what extent does microenvironment impose "stemness" on cells? Are damaged stem cells replenished by pre-existing stem cells or can more differentiated progeny acquire "stemness"? These issues are at the heart of what a stem cell is. Thus these advances should allow us to explore the molecular basis of cell dedifferentiation and reprogramming and to answer fundamental questions about tissue adaptive responses. PROJECT NARRATIVE: Stem cells are present in many tissues and maintain homeostasis of those tissues throughout life, and they are believed to be the major contributors to tissue regeneration following injury or transplantation. The goal of this project is to improve our understanding of stem cells biology for reparative and regenerative purposes.